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X-ray radiolytic labeling uses broadband X-rays for in situ hydroxyl radical labeling to map protein interactions and conformation. High flux density beams are essential to overcome radical scavengers. However, conventional sample delivery environments, such as capillary flow, limit the use of a fully unattenuated focused broadband beam. An alternative is to use a liquid jet, and we have previously demonstrated that use of this form of sample delivery can increase labeling by tenfold at an unfocused X-ray source. Here we report the first use of a liquid jet for automated inline quantitative fluorescence dosage characterization and sample exposure at a high flux density microfocused synchrotron beamline. Our approach enables exposure times in single-digit microseconds while retaining a high level of side-chain labeling. This development significantly boosts the method's overall effectiveness and efficiency, generates high-quality data, and opens up the arena for high throughput and ultrafast time-resolved in situ hydroxyl radical labeling., (© 2022. The Author(s).)

In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution is necessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments., (open access.)

Understanding multicomponent binding interactions in protein-ligand, protein-protein, and competition systems is essential for fundamental biology and drug discovery. Hand-deriving equations quickly become unfeasible when the number of components is increased, and direct analytical solutions only exist to a certain complexity. To address this problem and allow easy access to simulation, plotting, and parameter fitting to complex systems at equilibrium, we present the Python package PyBindingCurve. We apply this software to explore homodimer and heterodimer formations culminating in the discovery that under certain conditions, homodimers are easier to break with an inhibitor than heterodimers and may also be more readily depleted. This is a potentially valuable and overlooked phenomenon of great importance to drug discovery. PyBindingCurve may be expanded to operate on any equilibrium binding system and allows definition of custom systems using a simple syntax. PyBindingCurve is available under the MIT license at https://github.com/stevenshave/pybindingcurve as the Python source code accompanied by examples and as an easily installable package within the Python Package Index.

The self-assembly of biomacromolecules is an extremely important process. It is potentially useful in the fields of life science and materials science. To carry out the study on the self-assembly of proteins, it is necessary to find out the suitable self-assembly conditions, which have always been a challenging task in practice. Inspired by the screening technique in the field of protein crystallization, we proposed using the same screening technique for seeking suitable protein self-assembly conditions. Based on this consideration, we selected 5 proteins (β-lactoglobulin, hemoglobin, pepsin, lysozyme, α-chymotrypsinogen (II) A) together with 5 screening kits (Index TM , BML, Morpheus, JCSG, PEG/Ion Screen TM ) to investigate the performance of these crystallization screening techniques in order to discover new optimized conditions of protein self-assembly. The screens were all kept at 293 K for certain days, and were analyzed using optical microscope, scanning electron microscope, transmission electron microscope, atomic force microscope, fluorescence microscope, and atomic absorption spectroscope. The results demonstrated that the method of protein crystallization screening can be successfully applied in the screening of self-assembly conditions. This method is fast, high throughput, and easily implemented in an automated system, with a low protein consumption feature. These results suggested that such strategy can be applied to finding new conditions or forms in routine research of protein self-assembly. KEY POINTS: • Protein crystallization screening method is successfully applied in the screening of self-assembly conditions. • This screening method can be applied on various kinds of proteins and possess a feature of low protein consumption. • This screening method is fast, high throughput, and easily implemented in an automated system.

Objective: To investigate the effect of silencing LNK gene on the expression of EPO and EPOR in acute myeloid leukemia cells (THP-1)., Methods: THP-1 cells were cultured. The lentivirus was used as a vector to silence the LNK gene stably. After 72 hours of infection, GFP expression level was detected by the fluorescent inverted microscopy. The lentiviral Infection efficiencies were monitored by flow cytometry. The LNK silencing effect was confirmed. The mRNA expressions of EPO and EPOR were detected by RT-PCR. The protein levels of LNK, EPO and EPOR were detected by Western blot., Results: At the time-point of 72 hours after lentivirus infection, the expression level of GFP was above 85% detected by fluorescent inverted microscopy. The infection efficiency was above 99% by flow cytometry. mRNA expressions of LNK, EPO and EPOR in LNK silencing group were signifycantly lower than those in control group (P＜0.05). The protein levels of LNK, EPO and EPOR in LNK silencing group were significantly lower than those in the control group (P＜0.05)., Conclusion: THP-1 cell line of LNK gene silencing has been successfully established,the LNK gene has been silenced, the expression of EPO and EPOR decrease, indicating that LNK may participate in the regulation of EPO and EPOR.

Protein S-nitrosylation (SNO) is a typical reversible, redox-dependent and post-translational modification that involves covalent modification of cysteine residues with nitric oxide (NO) for the thiol group. Numerous experiments have shown that SNO plays a major role in cell function and pathophysiology. In order to rapidly analysis the big sets of data, the computing methods for identifying the SNO sites are being considered as necessary auxiliary tools. In this study, multiple features including Parallel correlation pseudo amino acid composition (PC-PseAAC), Basic kmer1 (kmer1), Basic kmer2 (kmer2), General parallel correlation pseudo amino acid composition (PC-PseAAC_G), Adapted Normal distribution Bi-Profile Bayes (ANBPB), Double Bi-Profile Bayes (DBPB), Bi-Profile Bayes (BPB), Incorporating Amino Acid Pairwise (IAAPair) and Position-specific Tri-Amino Acid Propensity(PSTAAP) were employed to extract the sequence information. To remove information redundancy, information gain (IG) was applied to evaluate the importance of amino acids, which is the information entropy of class after subtracting the conditional entropy for the given amino acid. The prediction performance of the SNO sites was found to be best by using the cross-validation and independent tests. In addition, we also calculated four commonly used performance measurements, i.e. Sensitivity (Sn), Specificity (Sp), Accuracy (Acc), and the Matthew's Correlation Coefficient (MCC). For the training dataset, the overall Acc was 83.11%, the MCC was 0.6617. For an independent test dataset, Acc was 73.17%, and MCC was 0.3788. The results indicate that our method is likely to complement the existing prediction methods and is a useful tool for effective identification of the SNO sites.

Regulated local translation-whereby specific mRNAs are transported and localized in subcellular domains where they are translated in response to regional signals-allows for remote control of gene expression to concentrate proteins in subcellular compartments. Neurons are highly polarized cells with unique features favoring local control for axonal pathfinding and synaptic plasticity, which are key processes involved in constructing functional circuits in the developing brain. Neurodevelopmental disorders are caused by genetic or environmental factors that disturb the nervous system's development during prenatal and early childhood periods. The growing list of genetic mutations that affect mRNA translation raises the question of whether aberrant translatomes in individuals with neurodevelopmental disorders share common molecular features underlying their stereotypical phenotypes and, vice versa, cause a certain degree of phenotypic heterogeneity. Here, we briefly give an overview of the role of local translation during neuronal development. We take the autism-risk gene list and discuss the molecules that (perhaps) are involved in mRNA transport and translation. Both exaggerated and suppressed translation caused by mutations in those genes have been identified or suggested. Finally, we discuss some proof-of-principle regimens for use in autism mouse models to correct dysregulated translation., (© 2018 Wiley Periodicals, Inc.)

Congenital muscular dystrophy type 1C (MDC1C) is caused by the homozygous or compound heterozygous mutations of the FKRP gene. This article reported the clinical and mutation features of a child with MDC1C. The boy aged 8 months visited the hospital due to delayed development. As for clinical manifestations, the boy could not turn over or sit stably by himself, and there was a significant reduction in muscle tension; biceps reflex in both upper extremities and patellar tendon reflex and Achilles tendon reflex in both lower extremities could not be induced. The boy also had a stereotyped facial expression and strabismus. Gene detection revealed c.350C>G and c.1303C>T compound heterozygous mutations in the FKRP gene. The c.350C>G mutation came from the mother and had been reported as a pathogenic missense mutation. The c.1303C>T mutation came from the father and was a new missense mutation, and a bioinformatics analysis showed that it might be a pathogenic mutation. The boy was diagnosed with MDC1C with reference to the clinical features of hypotonia and motor developmental delay and FKRP gene mutation sequencing.

Shell matrix proteins (SMPs) have important functions in biomineralization. In the past decades, the roles of SMPs were gradually revealed. In 2015, our group identified 72 unique SMPs in Pinctada fucata, among which Alveoline-like (Alv) protein was reported to have homologous genes in Pinctada maxima and Pinctada margaritifera. In this study, the full-length cDNA sequence of Alv and the functional analysis of Alv protein during shell formation were explored. The deduced protein (Alv), which has a molecular mass of 24.9 kDa and an isoelectric point of 11.34, was characterized, and the functional analyses was explored in vivo and in vitro. The Alv gene has high expression in mantle and could response to notching damage. The functional inhibition of Alv protein in vivo by injecting recombinant Alv (rAlv) antibodies destroyed prism structure but accelerated nacre growth. Western blot and immunofluorescence staining showed that native Alv exists in the EDTA-insoluble matrix of both prismatic and nacreous layers and has different distribution patterns in the inner or outer prismatic layer. Taken together, the characterization and functional analyses of matrix protein Alv could expand our understanding of basic matrix proteins and their functions during shell formation.

Protein crystallization is sensitive to the environment, while audible sound, as a physical and environmental factor during the entire process, is always ignored. We have previously reported that protein crystallization can be affected by a computer-generated monotonous sound with fixed frequency and amplitude. However, real-world sounds are not so simple but are complicated by parameters (frequency, amplitude, timbre, etc.) that vary over time. In this work, from three sound categories (music, speech, and environmental sound), we selected 26 different sounds and evaluated their effects on protein crystallization. The correlation between the sound parameters and the crystallization success rate was studied mathematically. The results showed that the real-world sounds, similar to the artificial monotonous sounds, could not only affect protein crystallization, but also improve crystal quality. Crystallization was dependent not only on the frequency, amplitude, volume, irradiation time, and overall energy of the sounds but also on their spectral characteristics. Based on these results, we suggest that intentionally applying environmental sound may be a simple and useful tool to promote protein crystallization., (Copyright © 2018. Published by Elsevier B.V.)

Peptide mapping with liquid chromatography-tandem mass spectrometry (LC-MS/MS) is an important analytical method for characterization of post-translational and chemical modifications in therapeutic proteins. Despite its importance, there is currently no consensus on the statistical analysis of the resulting data. In this manuscript, we distinguish three statistical goals for therapeutic protein characterization: (1) estimation of site occupancy of modifications in one condition, (2) detection of differential site occupancy between conditions, and (3) estimation of combined site occupancy across multiple modification sites. We propose an approach, which addresses these goals in terms of summarizing the quantitative information from the mass spectra, statistical modeling, and model-based analysis of LC-MS/MS data. We illustrate the approach using an LC-MS/MS experiment from an antibody-drug conjugate and its monoclonal antibody intermediate. The performance was compared to a 'naïve' data analysis approach, by using computer simulation, evaluation of differential site occupancy in positive and negative controls, and comparisons of estimated site occupancy with orthogonal experimental measurements of N-linked glycoforms and total oxidation. The results demonstrated the importance of replicated studies of protein characterization, and of appropriate statistical modeling, for reproducible, accurate and efficient site occupancy estimation and differential analysis.

Background: We evaluated the performance of human epididymis protein 4 (HE4), cancer antigen 125(CA 125) and Risk of Ovarian Malignancy Algorithm (ROMA) in distinguishing between benign and malignant pelvic masses in Chinese women., Method: From April to December 2012, women with a pelvic mass scheduled to have surgery were enrolled in a prospective, multi-center study conducted in 5 different regions in China. Preoperative serum concentrations of HE4 and CA 125 were examined and ROMA was calculated., Results: A total of 684 women with a pelvic mass were included, of which 482 were diagnosed with benign conditions and 202 were diagnosed with malignant ovarian tumors. At cutoffs of 7.4% and 25.3% for ROMA, the sensitivities and specificities were 85.6% and 81.7% for all patients, 85.7% and 81.5% for premenopausal women, and 85.6% and 83.9% for postmenopausal women, respectively. The ROC-AUC of ROMA was significantly better than that of HE4 (P=0.0003) or CA 125 (P<0.0001) for all malignant diseases (including EOC, Non-EOC, LMP, metastases and other pelvic malignancy with no involvement of the ovaries) compared with benign diseases for all patients., Conclusions: We demonstrated the efficiency of ROMA in the distinction of ovarian cancers from benign disease in a multiple-regions Chinese population, especially in premenopausal women., (Copyright © 2017 Elsevier B.V. All rights reserved.)

Objective: To compare the mutation and single nucleotide polymorphism (SNP) of LNK gene between chronic myeloid leukemia(CML) and control groups, and to explore the relationship between LNK gene variation and the occurrence of CML., Methods: A total of 36 patients with CML were selected, 46 healthy persons were used as normal controls. DNA was extracted from bone marrow and peripheral blood, BCR/ABL1 fusion gene was detected by Q-PCR. The whole exon of LNK gene was amplified by PCR. The amplified sequences included the Rs3184504 (C/T) and Rs78894077 (A/C/G/T) affecting the expression of amino acids in LNK gene, and the Rs7973120 (A/T) unaffecting the expression of the amino acids. The mutations and SNP of LNK gene were analyzed by DNA sequencing., Results: Thirty-six cases of CML had BCR/ABL1 mutation, while no mutation was found in the control group. One case of CML had LNK heterozygous mutation (A300V), and the mutation rate was 2.8%, no mutation was seen in normal control group. Rs3184504: C/T allele frequency was 50%/50% in the control group, 94.4%/5.6% in the CML group, and the C allele in CML group was significantly higher than that in the control group; CC genotype accounted for 94.4% (P<0.01). Rs78894077: C/T allele in the control group was 9.8%/90.2%, in CML group was 16.7%/83.3%, the difference was not statistically significant(P>0.05); but CC genotype in CML group was very statistically significant higher than that in control group(P<0.01). Rs7973120: A/T allele frequency was 10.9%/89.1% in the control group, 25%/75% in the CML group, the LNK A allele in CML group was very significantly higher than that in control group (P<0.01)., Conclusion: CML patients have been confirmed to have LNK mutation; the SNPs of LNK are related with the development of CML, and the most CML patients carry the LNK Rs3184504 C allele and the Rs7973120 A allele.

Protein crystallization under a magnetic field is an interesting research topic because a magnetic field may provide a special environment to acquire improved quality protein crystals. Because high-quality protein crystals are very useful in high-resolution structure determination using diffraction techniques (X-ray, neutron, and electron diffraction), research using magnetic fields in protein crystallization has attracted substantial interest; some studies have been performed in the past two decades. In this research field, the hardware is especially essential for successful studies because the environment is special and the design and utilization of the research apparatus in such an environment requires special considerations related to the magnetic field. This paper reviews the hardware for protein crystallization (including the magnet systems and the apparatus designed for use in a magnetic field) and progress in this area. Future prospects in this field will also be discussed., Competing Interests: The authors declare no conflicts of interest.

Neuropathic pain, caused by a lesion or dysfunction of the somatosensory nervous system, is a severe debilitating condition with which clinical treatment remains challenging. Jun activation domain-binding protein (JAB1) is a multifunctional protein that participates in several signaling pathways, controlling cell proliferation and apoptosis. However, the expression and possible function of JAB1 in the pathogenesis of neuropathic pain has not been elucidated. This study aimed to investigate the possible involvement of JAB1. Here, employing a neuropathic pain model induced by chronic constriction injury (CCI) on rats, we reported the role of JAB1 in the maintenance of neuropathic pain. By western blot, we found that CCI markedly up-regulated JAB1 expression in the dorsal root ganglion (DRG) and spinal cord. Immunofluorescent assay demonstrated that JAB1 was extensively localized in IB4-, CGRP- and NF200-positive neurons in the injured L5 DRG, and mainly co-localized with NeuN in spinal cord. In addition, we showed that CCI induced phosphorylation of p65 and JNK in vivo. Intrathecal injection of JAB1 siRNA significantly attenuated the CCI-induced JNK and p65 phosphorylation and alleviated both mechanical allodynia and heat hyperalgesia in rats. Taken together, these results suggested that JAB1 promotes neuropathic pain via positively regulating JNK and NF-κB activation.

Objective: LNK is an adapter protein negatively regulating the JAK/STAT cell signaling pathway. In this study, we observed the correlation between variation in LNK gene and the clinical type of myeloproliferative neoplasms (MPN)., Methods: A total of 285 MPN cases were recruited, including essential thrombocythemia (ET) 154 cases, polycythemia vera (PV) 76 cases, primary myelofibrosis (PMF) 19 cases, and chronic myeloid leukemia (CML) 36 cases. Ninety-three healthy individuals were used as normal controls. V617F mutation in JAK2 was identified by allele-specific PCR method, RT-PCR was used for the detection of BCR/ABL1 fusion gene, and mutations and variations in coding exons and their flanking sequences of LNK gene were examined by PCR-sequencing., Results: Missense mutations of A300V, V402M, and R415H in LNK were found in 8 patients including ET (4 cases, all combined with JAK2-V617F mutation), PV (2 cases, one combined with JAK2-V617F mutation), PMF (one case, combined with JAK2-V617F mutation) and CML (one case, combined with BCR/ABL1 fusion gene). The genotype and allele frequencies of the three SNPs (rs3184504, rs111340708 and rs78894077) in LNK were significantly different between MPN patients and controls. For rs3184504 (T/C, in exon2), the T allele (p.262W) and TT genotype were frequently seen in ET, PV and PMF (P<0.01), and C allele (p.262R) and CC genotype were frequently seen in CML (P<0.01). For rs78894077 (T/C, in exon1), the T allele (p.242S) was frequently found in ET (P<0.05). For rs111340708 (TGGGGx5/TGGGGx4, in intron 5), the TGGGG x4 allele was infrequently found in ET, PMF and CML(P<0.01)., Conclusion: Mutations in LNK could be found in some of MPN patients in the presence or absence of JAK2-V617F mutation. Several polymorphisms in LNK gene may affect the clinical type or the genetic predisposition of MPN.

Dual thermo- and pH-responsive chromatography has been proposed using poly(N-isopropylacrylamide-co-butyl methacrylate-co-N,N-dimethylaminopropyl acrylamide) (P(NIPAM-co-BMA-co-DMAPAAM)) brushes grafted gigaporous polystyrene microspheres (GPM) as matrix. Atom transfer radical polymerization (ATRP) initiator was first coupled onto GPM through Friedel-Crafts acylation with 2-bromoisobutyryl bromide. The dual-responsive polymer brushes were then grafted onto GPM via surface-initiated ATRP. The surface composition, gigaporous structure, protein adsorption and dual-responsive chromatographic properties of the matrix (GPM-P(NIPAM-co-BMA-co-DMAPAAM) were characterized in detail. Results showed that GPM were successfully grafted with thermoresponsive cationic polymer brushes and that the gigaporous structure was well maintained. A column packed with GPM-P(NIPAM-co-BMA-co-DMAPAAM presented low backpressure, good permeability and appreciable thermo-responsibility. By changing pH of the mobile phase and temperature of the column in turn, the column can separate three model proteins at the mobile phase velocity up to 2528cmh(-1). A separation mechanism of this matrix was also proposed. All results indicate that the dual thermo- and pH-responsive chromatography matrix has great potentials in 'green' high-speed protein chromatography., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Increased emphasis on the development of biologics has placed a significant focus on anti-drug antibody (ADA) detection. To address this need, several immunoassay formats have been described for use in characterizing potential immune responses. Two commonly utilized methods include the affinity capture elution (ACE) and bridging formats. While these approaches have been effective in supporting many clinical initiatives, both possess potential disadvantages. Here, we compare these standard methods to a novel format that addresses these noted drawbacks., Results: A novel assay format has been designed to incorporate the benefits of the ACE and bridging methods while overcoming the disadvantages incurred with each approach. The described ACE-Bridge format exhibits excellent sensitivity and precision while providing superior drug tolerance when compared to bridging formats. Further, this assay format is not susceptible to the endogenous target interference that can be an issue in the ACE format., Conclusions: The ACE-Bridge format provides an often superior option as a screening method to monitor patient ADA responses. This method is unique in its ability to measure ADA in the presence of high circulating endogenous target concentrations (>100 ng/mL) while demonstrating very high drug tolerance., (Copyright © 2016 Elsevier B.V. All rights reserved.)

We demonstrate the use of cryogenic super-resolution correlative light and electron microscopy (csCLEM) to precisely determine the spatial relationship between proteins and their native cellular structures. Several fluorescent proteins (FPs) were found to be photoswitchable and emitted far more photons under our cryogenic imaging condition, resulting in higher localization precision which is comparable to ambient super-resolution imaging. Vitrified specimens were prepared by high pressure freezing and cryo-sectioning to maintain a near-native state with better fluorescence preservation. A 2-3-fold improvement of resolution over the recent reports was achieved due to the photon budget performance of screening out Dronpa and optimized imaging conditions, even with thin sections which is at a disadvantage when calculate the structure resolution from label density. We extended csCLEM to mammalian cells by introducing cryo-sectioning and observed good correlation of a mitochondrial protein with the mitochondrial outer membrane at nanometer resolution in three dimensions.

Protein crystallization is affected by many parameters, among which certain parameters have not been well controlled. The temperature at which the protein and precipitant solutions are mixed (i.e., the ambient temperature during mixing) is such a parameter that is typically not well controlled and is often ignored. In this paper, we show that this temperature can influence protein crystallization. The experimental results showed that both higher and lower mixing temperatures can enhance the success of crystallization, which follows a parabolic curve with an increasing ambient temperature. This work illustrates that the crystallization solution preparation temperature is also an important parameter for protein crystallization. Uncontrolled or poorly controlled room temperature may yield poor reproducibility in protein crystallization.

A high-speed thermoresponsive medium was developed by grafting poly(N-isopropylacrylamide-co-butyl methacrylate) (P(NIPAM-co-BMA)) brushes onto gigaporous polystyrene (PS) microspheres via surface-initiated atom transfer radical polymerization (ATRP) technique, which has strong mechanical strength, good chemical stability and high mass transfer rate for biomacromolecules. The gigaporous structure, surface chemical composition, static protein adsorption, and thermoresponsive chromatographic properties of prepared medium (PS-P(NIPAM-co-BMA)) were characterized in detail. Results showed that the PS microspheres were successfully grafted with P(NIPAM-co-BMA) brushes and that the gigaporous structure was robustly maintained. After grafting, the nonspecific adsorption of proteins on PS microspheres was greatly reduced. A column packed with PS-P(NIPAM-co-BMA) exhibited low backpressure and significant thermo-responsibility. By simply changing the column temperature, it was able to separate three model proteins at the mobile phase velocity up to 2167 cm h(-1). In conclusion, the thermoresponsive polymer brushes grafted gigaporous PS microspheres prepared by ATRP are very promising in 'green' high-speed preparative protein chromatography., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Scaffolds that can achieve cell adhesion and controlled release of protein drugs are very promising in bone tissue engineering. Due to their biocompatibility and injectablity, poly(lactide-co-glycolide acid) (PLGA) porous microspheres (PLGA-pMS) present potential scaffolds in bone tissue engineering. However, their application is hampered by the burst release of protein drugs and hydrophobicity that leads to poor cell adhesion. To overcome these drawbacks, we developed novel PLGA-pMS by incorporating bovine serum albumin (BSA) loaded chitosan microspheres (CS-MS) in Gly-Arg-Gly-Asp-Ser-Pro-Cys (GRGDSPC) modified PLGA-pMS (CS-MS/PLGA-pMS). GRGDSPC was used to enhance the hydrophilicity and cell affinity of the porous microspheres. Results showed that PLGA-pMS had a size of 446.77±19.46μm, with an average surface pore size of 21.56±3.02μm, whereas CS-MS had a size of 15.98±0.96μm and 16.35±0.38μm (5% and 10% TPP-prepared CS-MS, respectively). A scanning electron microscope (SEM) and a confocal laser scanning microscope (CLSM) revealed that CS-MS were partly embedded in the PLGA matrices and the integrity of CS-MS was retained. Thermogravimetry analyzer (TGA) also demonstrated that CS-MS were incorporated into PLGA-pMS. The CS-MS/PLGA-pMS had a size of 454.02±16.09μm, with a BSA encapsulation efficiency of 53.19±1.67% and 62.16±3.44% (5% and 10% TPP-prepared CS-MS, respectively). CS-MS/PLGA-pMS exhibited a sustained FITC-BSA release for 28 days. Modification of GRGDSPC significantly improved adhesion of MG-63 cells on the porous microspheres. In conclusion, CS-MS/PLGA-pMS may act as potential bifunctional scaffolds for bone tissue engineering., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Cardiomyocyte cell division and replication in mammals proceed through embryonic development and abruptly decline soon after birth. The process governing cardiomyocyte cell cycle arrest is poorly understood. Here we carry out whole-exome sequencing in an infant with evidence of persistent postnatal cardiomyocyte replication to determine the genetic risk factors. We identify compound heterozygous ALMS1 mutations in the proband, and confirm their presence in her affected sibling, one copy inherited from each heterozygous parent. Next, we recognize homozygous or compound heterozygous truncating mutations in ALMS1 in four other children with high levels of postnatal cardiomyocyte proliferation. Alms1 mRNA knockdown increases multiple markers of proliferation in cardiomyocytes, the percentage of cardiomyocytes in G2/M phases, and the number of cardiomyocytes by 10% in cultured cells. Homozygous Alms1-mutant mice have increased cardiomyocyte proliferation at 2 weeks postnatal compared with wild-type littermates. We conclude that deficiency of Alström protein impairs postnatal cardiomyocyte cell cycle arrest.

This chapter presents an overview of quantitative fluorescence brightness experiments with special emphasis on single-color measurements of protein homo-interactions inside living cells. We discuss practical considerations in the choice of the fluorescent labels and the calibration measurements necessary for quantitative interpretation of brightness experiments. Methods to identify and avoid common pitfalls, such as bleaching and saturation, are addressed. We examine the interpretation of brightness data with moment analysis. In particular, we focus on how to avoid or correct for undersampling, as well as how to characterize and adjust for photon detector effects. We conclude by describing brightness titration experiments which determine the binding curve and stoichiometry of a protein from apparent brightness data.

High-quality crystals are key to obtaining accurate three-dimensional structures of proteins using X-ray diffraction techniques. However, obtaining such protein crystals is often a challenge. Several containerless crystallization techniques have been reported to have the ability to improve crystal quality, but it is unknown which is the most favourable way to grow high-quality protein crystals. In this paper, a quality comparison of protein crystals which were grown under three containerless conditions provided by diamagnetic levitation, silicone oil and agarose gel was conducted. A control experiment on a vessel wall was also simultaneously carried out. Seven different proteins were crystallized under the four conditions, and the crystal quality was assessed in terms of the resolution limit, the mosaicity and the Rmerge. It was found that the crystals grown under the three containerless conditions demonstrated better morphology than those of the control. X-ray diffraction data indicated that the quality of the crystals grown under the three containerless conditions was better than that of the control. Of the three containerless crystallization techniques, the diamagnetic levitation technique exhibited the best performance in enhancing crystal quality. This paper is to our knowledge the first report of improvement of crystal quality using a diamagnetic levitation technique. Crystals obtained from agarose gel demonstrated the second best improvement in crystal quality. The study indicated that the diamagnetic levitation technique is indeed a favourable method for growing high-quality protein crystals, and its utilization is thus potentially useful in practical efforts to obtain well diffracting protein crystals.

Myeloproliferative neoplasms ( MPN ) is a class of clonal hematopoietic stem cell disease. Studies found that the JAK-STAT signaling pathway is closely related to the pathogenesis of MPN. The lymphocyte-specific adaptor protein (LNK) gene negatively regulates Janus kinase-signal transducer and activator of transcription (JAK-STAT) signaling and may play an important role in the pathogenesis of MPN. Especially in JAK2 mutation-negative MPN, LNK gene specific mutations may be the key to cause MPN subtypes. Certain single nucleotide polymorphism of LNK gene regulation of hematopoietic cells in different directions may also be important influence factors of MPN performance for different subtypes. LNK gene functional changes lead to abnormal activation of the JAK-STAT signaling pathway, and may be a new mechanism of MPN. In this review, the role of LNK gene in MPN pathogenesis is briefly summarized.

In hepatocellular carcinomas (HCCs), the levels of histone H3 dimethylation at lysine 9 (H3K9me2) and its corresponding histone methyltransferase G9a are significantly elevated. Recently, G9a was reported to form a complex with the H3K9 methylation effector protein CDYL, but little is known about the expression of CDYL in HCC patients. The human CDYL gene produces two transcripts, a long form (CDYLa) and a short form (CDYLb), but it is unclear whether the protein products have different functions. The aim of this study was to investigate the distinctions between CDYLa and CDYLb and their expression levels in HCC tissues. We first examined binding abilities of the different CDYL forms with methylated H3 peptides by a pull-down assay. Human CDYLb (h-CDYLb) specifically recognized H3Kc9me2 and H3Kc9me3 modifications, whereas human CDYLa (h-CDYLa) did not interact with any methylated H3 peptides. Similarly, mouse CDYLb (m-CDYLb) specifically bound with di- and tri-methylated H3Kc9 peptides, while mouse CDYLa (m-CDYLa) lacked that ability. Affinity purification also was used to identify the distinct composition of the h-CDYLa or h-CDYLb protein complex. h-CDYLb was found in a multiprotein complex with G9a and GLP, while the h-CDYLa complex did not contain these two enzymes. Consistent with the protein complex composition, h-CDYLb and G9a were both upregulated in HCC tissues, compared with adjacent non-cancerous liver tissues. Furthermore, the positive correlation between expression levels of h-CDYLb and G9a was statistically significant. In contrast, h-CDYLa showed no enrichment in HCC tissues. These findings suggest that h-CDYLb and G9a are cooperatively involved in HCC., (Copyright © 2013 Wiley Periodicals, Inc.)

The pH of a solution is an important parameter in crystallization that needs to be controlled in order to ensure success. The actual pH of the crystallization droplet is determined by the combined contribution of the buffers in the screening and protein solutions, although the contribution of the latter to the pH is often ignored. In this study, the effects of the buffer and protein solution pH values on the results of screening are systematically investigated. It was found that these parameters significantly affected the results and thus the following strategy for the selection of appropriate pH values is proposed: (i) when screening with only one protein solution, the pH should be as low, as high or as divergent from the pI as possible for a basic, acidic or neutral protein, respectively, within its stable pH range; (ii) when screening with two protein solutions, the pH values should be well separated from one another; and (iii) when multiple pH values are utilized, an even distribution of pH values is the best approach to increase the success rate of crystallization.

Most protein crystallisation begins from heterogeneous nucleation; in practice, crystallisation typically occurs in the presence of a solid surface in the solution. The solid surface provides a nucleation site such that the energy barrier for nucleation is lower on the surface than in the bulk solution. Different types of solid surfaces exhibit different surface energies, and the nucleation barriers depend on the characteristics of the solid surfaces. Therefore, treatment of the solid surface may alter the surface properties to increase the chance to obtain protein crystals. In this paper, we propose a method to modify the glass cover slip using a self-assembled monolayer (SAM) of functional groups (methyl, sulfydryl and amino), and we investigated the effect of each SAM on protein crystallisation. The results indicated that both crystallisation success rate in a reproducibility study, and crystallisation hits in a crystallisation screening study, were increased using the SAMs, among which, the methyl-modified SAM demonstrated the most significant improvement. These results illustrated that directly modifying the crystallisation plates or glass cover slips to create surfaces that favour heterogeneous nucleation can be potentially useful in practical protein crystallisation, and the utilisation of a SAM containing a functional group can be considered a promising technique for the treatment of the surfaces that will directly contact the crystallisation solution.

Brightness analysis provides a powerful tool for the study of protein interactions both in solution and in living cells. We provide a brief survey of some widely used techniques for extracting brightness from fluorescent fluctuation spectroscopy experiments. While all the techniques are equivalent under ideal conditions, we touch upon their relative strengths and discuss in detail a specific scenario wherein the photon-counting histogram (PCH) separates the brightness of rare, bright particles from a dominant background. In a practical vein for ensuring quantitative and unbiased brightness data, we address a number of potential issues stemming from both theoretical assumptions and experimental realities. Two additional issues arising from geometry are examined in greater detail. An oil-immersion objective skews the geometry of the excitation volume as a function of penetration depth. The bias can be characterized and corrected or avoided through the use of a water-immersion objective. Brightness measurements in thin sample geometries, frequently encountered in cells, may be biased. We use z-scan FFS to characterize sample geometry and correct any resulting bias in the brightness., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Although the crustacean Artemia has been commonly used as an experimental organism and served as a live bait feed for aquaculture, gene transfer system on Artemia sp. to generate stable lines is not well developed. In this study, we optimized a condition for cyst-eletroporation and generated stable lines of transgenic A. sinica. Two expression plasmids directed by the hybrid promoters of cytomegalovirus (CMV) and medaka β-actin (Mβ) were co-electroporated on decapsulated cysts: pCMV-Mβ-GFP contained GFP reporter gene and pCMV-Mβ-ypGH contained yellowfin porgy GH (ypGH) cDNA. We examined the GFP shown in the Artemia larvae and found that the expression rate was 13.3% (3,219 out of 24,054 examined). We then chose 200 G0 founders which strongly expressed GFP to generate transgenic lines. Homozygotic strains derived from F4 generation of each transgenic line, A3 and A8, were obtained. We proved that transgenic lines A3 and A8 also harbored pCMV-Mβ-ypGH and produced recombinant ypGH with a concentration of 0.089 and 0.032 μg per 50 homozygotic nauplii, respectively. Ten live Artemia nauplii were fed daily to zebrafish larvae during 25 to 35 days of post-fertilization. The average body length gain rates of zebrafish larvae fed transgenic Artemia were 16-20% greater than those of control group, indicating the exogenous ypGH produced by transgenic Artemia is functional. Therefore, we concluded that the transgenesis on Artemia is developed, and transgenic Artemia might be highly potentially useful as a new bioreactor material for application in aquaculture and biological researches.

The nucleation zone has to be reached for any crystal to grow, and the search for crystallization conditions of new proteins is a trial and error process. Here a convenient screening strategy is studied in detail that varies the volume ratio of protein sample to the reservoir solution in the drop to initiate crystallization that is named "composition modification". It is applied after the first screen and has been studied with twelve proteins. Statistical analysis shows a significant improvement in screening using this strategy. The average improvement of "hits" at different temperatures is between 32 and 42%, for examples, 41.8% ± 14.0% and 35.7% ± 12.4% (± standard deviation) at 288 K and 300 K, respectively. Remarkably, some new crystals were found by composition modification which increased the probability of reaching the nucleation zone to initiate crystallization. This was confirmed by a phase diagram study. It is also demonstrated that composition modification can further increase crystallisation success significantly (1.3 times) after the improvement of "hits" by temperature screening. The trajectories of different composition modifications during vapour diffusion were plotted, further demonstrating that protein crystallizability can be increased by hitting more parts of the nucleation zone. It was also found to facilitate the finding of initial crystals for proteins of low solubility. These proteins gradually become more concentrated during the vapour diffusion process starting from a larger protein solution ratio in the initial mixture.

Characterization of bright particles at low concentrations by fluorescence fluctuation spectroscopy (FFS) is challenging, because the event rate of particle detection is low and fluorescence background contributes significantly to the measured signal. It is straightforward to increase the event rate by flow, but the high background continues to be problematic for fluorescence correlation spectroscopy. Here, we characterize the use of photon-counting histogram analysis in the presence of flow. We demonstrate that a photon-counting histogram efficiently separates the particle signal from the background and faithfully determines the brightness and concentration of particles independent of flow speed, as long as undersampling is avoided. Brightness provides a measure of the number of fluorescently labeled proteins within a complex and has been used to determine stoichiometry of protein complexes in vivo and in vitro. We apply flow-FFS to determine the stoichiometry of the group specific antigen protein within viral-like particles of the human immunodeficiency virus type-1 from the brightness. Our results demonstrate that flow-FFS is a sensitive method for the characterization of complex macromolecular particles at low concentrations., (Copyright © 2010 Biophysical Society. Published by Elsevier Inc. All rights reserved.)

A single fluorescently labeled protein generates a short burst of light whenever it passes through a tiny observation volume created within a biological cell. The average amplitude of the burst is related to the stoichiometry of the fluorescently labeled protein complex. Fluorescence fluctuation spectroscopy quantifies the burst amplitude by introducing the brightness parameter. Brightness provides a spectroscopic marker for observing protein interactions and their stoichiometry directly inside cells. Not all fluorescent proteins are suitable for brightness experiments. Here we discuss how brightness properties of the fluorophore influence brightness measurements and how to identify a well-behaved fluorescent protein. Protein interactions and stoichiometry are determined from a brightness titration. Experimental details of brightness titration measurements are described together with the necessary calibration and control experiments., (Copyright 2010 Elsevier Inc. All rights reserved.)

Aim: To investigate the effect of betulinic acid (BA) on the proliferation, apoptosis and cell cycle of gastric adenocarcinoma cell AGS in vitro and the underlying mechanism., Methods: The effect of BA on the proliferation of AGS cells was measured by using 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium (MTT) assay. Apoptosis was analyzed by using Annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) double-labeled flow cytometry (FCM) and Hoechst 33258 staining. The influence of BA on cell cycle of AGS cells was tested by PI staining. Both FCM and reverse transcription-PCR (RT-PCR) technologies were applied to detect the expression of Hiwi and Cyclin B1., Results: BA exhibited significant cell proliferation inhibition, as well as its potency of inducing apoptosis in AGS cells in vitro in a time- and dose-dependent manner. The IC(50) value for 24 h was 18.25 microg/mL (95% confidence interval: 15.16 to 27.31 microg/mL). Cells treated with BA showed increased cell population in G(2)/M phase, with decreased S phase population. The expression of Hiwi and Cyclin B1 was down-regulated in BA-treated AGS cells in a dose-dependent manner., Conclusion: BA exerted potent effect on growth inhibition, G(2)/M cell cycle arrest and induction of apoptosis in AGS cells in vitro, possibly associated with the down-regulation of Hiwi and its downstream target Cyclin B1 expression. The potent antitumor capacity of BA suggested that it could be a promising new experimental anticancer agent in human gastric adenocarcinoma treatment.

Although protein prenylation is widely studied, there are few good methods for isolating prenylated proteins from their nonprenylated relatives. We report that crosslinked agarose (e.g., Sepharose) chromatography medium that has been chemically functionalized with beta-cyclodextrin (beta-CD) is extremely effective in affinity chromatography of prenylated proteins. In this study, a variety of proteins with C-terminal prenylation target ("CAAX box") sequences were enzymatically prenylated in vitro with natural and nonnatural prenyl diphosphate substrates. The prenylated protein products could then be isolated from starting materials by gravity chromatography or fast protein liquid chromatography (FPLC) on a beta-CD-Sepharose column. One particular prenylation reaction, farnesylation of an mCherry-CAAX fusion construct, was studied in detail. In this case, purified farnesylated product was unambiguously identified by electrospray mass spectrometry. In addition, when mCherry-CAAX was prenylated with a nonnatural, functional isoprenoid substrate, the functional group was maintained by chromatography on beta-CD-Sepharose, such that the resulting protein could be selectively bound at its C terminus to complementary functionality on a solid substrate. Finally, beta-CD-Sepharose FPLC was used to isolate prenylated mCherry-CAAX from crude HeLa cell lysate as a model for purifying prenylated proteins from cell extracts. We propose that this method could be generally useful to the community of researchers studying protein prenylation.

The purpose of this study was to examine the effect of charge ratio on the formation and properties of the chitosan (CS)-dextran sulfate (DS) nanoparticles developed for the delivery of water-soluble small and large molecules, including proteins. Rhodamine 6G (R6G) and bovine serum albumin (BSA) were chosen as model molecules. CS-DS nanoparticles were formulated by a complex coacervation process under mild conditions. The influence of formulation and process variables, including the charge ratio of the 2 ionic polymers, on particle size, zeta potential, and nanoparticle entrapment of R6G and BSA was studied. The in vitro release of R6G and BSA was also evaluated, and the integrity of BSA in the release fraction was assessed using sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Depending on the concentration and charge ratio of DS and CS, nanoparticles with varied size (>or=244 nm) and zeta potential (-47.1-60 mV) were obtained. High entrapment efficiency (98%) was achieved for both R6G and BSA when the charge ratio of the 2 ionic polymers was greater than 1.12. The release of both R6G and BSA from nanoparticles was based on the ion-exchange mechanism. BSA showed much slower continuous release for up to 7 days while still maintaining its integrity for an extended period. The CS-DS nanoparticles developed based on the modulation of charge ratio show promise as a system for controlled delivery of both small and large molecules, including proteins.

DNA fragmentation into internucleosomal fragments is the best recognized biochemical event of apoptosis. Two major caspase pathways have been identified in the signal transduction leading to DNA fragmentation: the receptor pathway and the mitochondrial pathway. DNA fragmentation factor (DFF) has been identified as a major apoptotic endonuclease in the internucleosomal DNA fragmentation process. However, the potential roles of caspases and DFF in internucleosomal DNA fragmentation induced by specific stimuli still need to be investigated since caspase-independent pathways and nuclease(s) other than DFF also play important roles during this process. In the present study, we investigated the activity of GP7 (4-[4"-(2",2",6",6"-tetramethyl-l"-piperidinyloxy) amino]-4'-demethyl epipodophyllotoxin), a new spin-labeled derivative of podophyllotoxin semi-synthesized by our university, to induce apoptosis of the human leukemia cell line NB4. GP7 induced the release of cytochrome-c from mitochondria, activations of caspase-3, -8, and -9, cleavage of DFF45/inhibitor of caspase-activated DNase, activation of DFF40/caspase-activated DNase, and apoptotic DNA fragmentation in NB4 cells. The broad-spectrum caspase inhibitor zVAD-fmk abrogated GP7-induced caspase-3, -8, and -9 activations but could not inhibit GP7-induced apoptotic DNA fragmentation in NB4 cells. Our findings suggest that GP7-induced apoptotic DNA fragmentation in NB4 cells is independent of caspase activation and DFF, although they are closely involved in this process.

Aim: To observe the effects of augmenter of liver regeneration (ALR) on Kupffer cells and to determine whether ALR promotes hepatocyte proliferation induced by Kupffer cells., Methods: Kupffer cells and hepatocytes were cultured in vitro and various concentrations of recombinant rat ALR (rrALR) were added. 3H-thymidine, BrdU and 3H-leucine incorporation was determined in cultured Kupffer cells and hepatocytes, in hepatocytes conditioned by Kupffer cells, and in associated medium. rrALR was labeled by iodination and used to determine its binding activity by Scatchard analysis in Kupffer cells and primarily cultured rat hepatocytes., Results: rrALR stimulated DNA replication in Kupffer cells and protein synthesis both in cells and in medium in a non-concentration-dependent manner. The effect was significant at the concentration of 1 microg/L ALR. However, rrALR had no effect on primarily cultured hepatocytes, when hepatocytes were cultured with the Kupffer cell medium conditioned by ALR, DNA replication and protein synthesis in hepatocytes increased significantly at the concentration of 1 microg/L ALR. When the ALR concentration was increased, its effect on hepatocyte proliferation decreased to the basal level. Scatchard analysis indicated the presence of a single class of high affinity receptors with a dissociation constant (Kd) of 0.883 nmol/L and a maximum binding capacity (Bmax) of 126.1 pmol/g protein in the rat Kupffer cells., Conclusion: ALR can promote hepatocyte proliferation induced by Kupffer cells, which is associated with the concentration of ALR, suggesting that Kupffer cells play a dual role in liver regeneration.

Background: The process of arteriogenesis after occlusion of a major artery is poorly understood. We have used high-resolution microcomputed tomography (mu-CT) imaging to define the arteriogenic response in the mouse model of hindlimb ischemia and to examine the effect of placental growth factor-1 (PlGF-1) on this process., Methods and Results: After common femoral artery ligation, mu-CT imaging demonstrated formation of collateral vessels originating near the ligation site in the upper limb and connecting to the ischemic calf muscle region. Three-dimensional mu-CT and quantitative image analysis revealed changes in the number of segments and the segmental volume of vessels, ranging from 8 to 160 microm in diameter. The medium-size vessels (48 to 160 microm) comprising 85% of the vascular volume were the major contributor (188%) to the change in vascular volume in response to ischemia. Intramuscular injections of Ad-PlGF-1 significantly increased Sca1+ cells in the circulation, alpha-actin-stained vessels, and perfusion of the ischemic hindlimb. These effects were predominantly associated with an increase in vascular volume contributed by the medium-size (96 to 144 microm) vessels as determined by mu-CT., Conclusions: High-resolution mu-CT delineated the formation of medium-size collaterals representing a major vascular change that contributed to the restoration of vascular volume after ischemia. This effect is selectively potentiated by PlGF-1. Such selective enhancement of arteriogenesis by therapeutically administered PlGF-1 demonstrates a desirable biological activity for promoting the growth of functionally relevant vasculature.

Fluorescence correlation spectroscopy is a potentially powerful tool for measuring protein-protein interactions directly in single living cells. We previously reported on the detection of homodimer formation in cells using molecular brightness analysis. Here, we extend the technique to detect binding between different proteins. Proteins are labeled with the fluorescent markers YFP and CFP. We first determine the coexpression ratio of both proteins by measuring the intensity ratio with a dual-color setup. The effect of fluorescence resonance energy transfer on the intensity ratio is explicitly taken into account. The brightness of cells coexpressing both proteins is measured in a single-color setup. Selecting the laser wavelength of the two-photon light source allows us to either coexcite both proteins or to selectively excite YFP-labeled proteins. This approach enables us to distinguish between homodimer and heterodimer formation. We first present the theory and then demonstrate experimental feasibility using the ligand binding domains of retinoic acid receptor (RARLBD) and of retinoid X receptor (RXRLBD). Both proteins form heterodimers, and RXRLBD also forms homodimers in the presence of its agonist. We explore binding between these proteins in the presence and absence of RXR agonist. Our results demonstrate that brightness analysis offers a quantitative method for determining protein interactions in cells.

Aim: To investigate the effect of cholesterol (Ch) on the growth and functional protein expression of rabbit bile duct fibroblasts., Methods: The cultured bile duct fibroblasts were divided randomly into two groups: the control group and the experiment group (fibroblasts were incubated respectively with 0.6 g/L Ch for 12, 24, 36 and 48 h). The growth and DNA synthesis of bile duct fibroblasts were measured by the means of (3)H-TdR incorporation. The total protein content of fibroblast was measured by BSA protein assay reagent kit, then the expression of alpha-actin was analyzed semi-quantitatively by Western blot., Results: After treatment with 0.6 g/L Ch for 12, 24, 36 and 48 h, the values of (3)H-TdR incorporation of bile duct fibroblasts were respectively 3.1+/-0.39, 3.8+/-0.37, 4.6+/-0.48 and 5.2+/-0.56 mBq/cell, and the values of the corresponding control groups were 3.0+/-0.33, 3.2+/-0.39, 3.7+/-0.49 and 4.3+/-0.43 mBq/cell. After comparing the values of experiment groups and their corresponding control groups, it was found that the (3)H-TdR incorporation of bile duct fibroblasts after treatment with 0.6 g/L Ch for 24, 36 and 48 h were significantly increased (P<0.05, P<0.01, P<0.01), while the (3)H-TdR incorporation of 12-h group was not different statistically from its control group. Ch had no obvious effect on the total protein content of fibroblasts. After incubated with 0.6 g/L Ch for 12, 24, 36 and 48 h, the total protein content of each experiment group was not altered markedly compared with its corresponding control group. The values of experiment groups were 0.246+/-0.051, 0.280+/-0.049, 0.263+/-0.044 and 0.275+/-0.056 ng/cell, and those of corresponding control groups were 0.253+/-0.048, 0.270+/-0.042, 0.258+/-0.050 and 0.270+/-0.045 ng/cell. Western blot analysis revealed that the alpha-actin expression in fibroblasts affected by Ch for 12 and 24 h was not markedly changed compared with their corresponding control groups (P>0.05), the values of total gray scale of 12- and 24-h groups were 1748+/-185 and 1756+/-173, respectively. But after stimulation with Ch for 36 h, the total gray scale of fibroblasts (1923+/-204) was significantly higher than that of control group (1734+/-197). When the time of Ch treatment was lengthened to 48 h, the alpha-actin expression was markedly elevated, the total gray scale was 2 189+/-231 (P<0.01 vs control group)., Conclusion: Moderately concentrated Ch can promote the proliferation of bile duct fibroblasts at early stage. With the prolongation of Ch treatment, the alpha-actin expression of fibroblasts was also increased, but the hypertrophy of fibroblasts was not observed.

To understand the expression and effect of mismatch repair genes, hMSH2 and hMLH1, and to investigate anti-leukemic cell proliferation mechanism of curcumin, the levels of both genes were detected by multiple comparative RT-PCR. The protein of hMSH2 was determined by flow cytometry (FCM) and the gene mutation of hMSH2 and hMLH1 were detected by PCR-SSCP and microsatellite instability assay. After UV irradiation, the gene expression of hMSH2 and hMLH1 was not increased and showed no response. This phenomenon was not ascribed to gene mutation, because PCP-SSCP and microsatellite instability assay revealed no abnormal gel-shift band in both genes. After irradiation and addition of curcumin, the expression of hMSH2 mRNA increased and the cellular apoptotic rate also increased at the same time. The difference was statistically significant as compared with groups without addition of curcumin and control groups (P < 0.05). Our results suggested that when MMR system was inhibited by the same agents, curcumin can remove this suppression and switch to cellular apoptosis.

Signal transduction of activin, one of the members in the transforming growth factor-beta superfamily, is initiated by ligand binding with two distinct membrane receptors (type II and type I) followed by activation of Smad2 or Smad3. We report here that activin-induced signaling is negatively regulated by another Smad molecule, Smad7. When expressed in Chinese hamster ovary cells, Smad7 inhibited the transcriptional response induced by either activin treatment or a constitutively active activin type I receptor (ALK-4). In addition, Smad7 also inhibited mouse FAST-2-mediated transactivation of the Xenopus Mix.2 promoter stimulated by the constitutively active ALK-4. Smad7 was able to directly associate with ALK-4 and this association was dependent on the phosphorylation of the type I receptor in its GS domain by activin type II receptors. Expression of kinase defective activin type II receptors decreased the association of Smad7 with ALK-4. Correspondingly, Smad7 bound poorly to a mutant ALK-4 bearing serine to alanine substitutions in four putative phosphorylation sites in its GS domain. These studies not only illustrated the counter regulatory function of Smad7 on activin signaling, but also indicated the involvement of phosphorylation at activin type I receptor in the inhibitory action of Smad7.

Plate-based proteomic sample preparation offers a solution to the large sample throughput demands in the biotechnology field where hundreds or thousands of engineered microbes are constructed for testing is routine. Meanwhile, sample preparation methods that work efficiently on broader microbial groups are desirable for new applications of proteomics in other fields, such as microbial communities. Here, we detail a step-by-step protocol that consists of cell lysis in an alkaline chemical buffer (NaOH/SDS) followed by protein precipitation with high-ionic strength acetone in 96-well format. The protocol works for a broad range of microbes (e.g., Gram-negative bacteria, Gram-positive bacteria, non-filamentous fungi) and the resulting proteins are ready for tryptic digestion for bottom-up quantitative proteomic analysis without the need for desalting column cleanup. The yield of protein using this protocol increases linearly with respect to the amount of starting biomass from 0.5-2.0 OD*mL of cells. By using a bench-top automated liquid dispenser, a cost-effective and environmentally-friendly option to eliminating pipette tips and reducing reagent waste, the protocol takes approximately 30 minutes to extract protein from 96 samples. Tests on mock mixtures showed expected results that the biomass composition structure is in close agreement with the experimental design. Lastly, we applied the protocol for the composition analysis of a synthetic community of environmental isolates grown on two different media. This protocol has been developed to facilitate rapid, low-variance sample preparation of hundreds of samples and allow flexibility for future protocol development.

The 5′-HOXD genes are important for chondrogenesis in vertebrates, but their roles in osteoarthritis (OA) are still ambiguous. In our study, 5′-HOXD genes involvement contributing to cartilage degradation and OA was investigated. In bioinformatics analysis of 5′-HOXD genes, we obtained the GSE169077 data set related to OA in the GEO and analyzed DEGs using the GEO2R tool attached to the GEO. Then, we screened the mRNA levels of 5′-HOXD genes by quantitative reverse transcriptase–polymerase chain reaction (qRT-PCR). We discovered that OA chondrocyte proliferation was inhibited, and apoptosis was increased. Moreover, it was discovered that SOX9 and COL2A1 were downregulated at mRNA and protein levels, while matrix metalloproteinases (MMPs) and a disintegrin-like and metalloproteinase with thrombospondin motifs (ADAMTSs) were upregulated. According to the results of differentially expressed genes (DEGs) and qRT-PCR, we evaluated the protein level of HOXD11 and found that the expression of HOXD11 was downregulated, reversed to MMPs and ADAMTSs but consistent with the cartilage-specific factors, SOX9 and COL2A1. In the lentivirus transfection experiments, HOXD11 overexpression reversed the effects in OA chondrocytes. In human OA articular cartilage, aberrant subchondral bone was formed in hematoxylin–eosin (H&E) and Safranin O and fast green (SOFG) staining results. Furthermore, according to immunohistochemistry findings, SOX9 and HOXD11 expression was inhibited. The results of this study established that HOXD11 was downregulated in OA cartilage and that overexpression of HOXD11 could prevent cartilage degradation in OA. [ABSTRACT FROM AUTHOR]

We examine the binding of fluorescent ligands to proteins by analyzing the fluctuation amplitude g(0) of fluorescence fluctuation experiments. The normalized variance g(0) depends on the molecular brightness and the concentration of each species in the sample. Thus a single g(0) measurement is not sufficient to resolve individual species. Titration of the ligand with protein establishes the link between molecular brightness and concentration by fitting g(0) to a binding model and allows the separation of species. We first apply g(0) analysis to binary dye mixtures with brightness ratios of 2 and 4 to demonstrate the feasibility of this technique. Next we consider the influence of binding on the fluctuation amplitude g(0). The dissociation coefficient, the molecular brightness ratio, and the stochiometry of binding strongly influence the fluctuation amplitude. We show that proteins with a single binding site can be clearly differentiated from proteins with two independent binding sites. The binding of fluorescein-labeled digoxigenin to a high-affinity anti-digoxin antibody was studied experimentally. A global analysis of the fluctuation amplitude and the fluorescence intensity not only recovered the dissociation coefficient and the number of binding sites, but also revealed the molecular heterogeneity of the hapten-antibody complex. Two species were used to model the molecular heterogeneity. We confirmed the molecular heterogeneity independently by fluorescence lifetime experiments, which gave fractional populations and molecular brightness values that were virtually identical to those of the g(0) analysis. The identification and characterization of molecular heterogeneity have far-reaching consequences for many biomolecular systems. We point out the important role fluctuation experiments may have in this area of research.